WO2020130166A1 - Composition comprenant un extrait de magnolia flos pour la prévention et le traitement de la rhinite allergique - Google Patents

Composition comprenant un extrait de magnolia flos pour la prévention et le traitement de la rhinite allergique Download PDF

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Publication number
WO2020130166A1
WO2020130166A1 PCT/KR2018/016074 KR2018016074W WO2020130166A1 WO 2020130166 A1 WO2020130166 A1 WO 2020130166A1 KR 2018016074 W KR2018016074 W KR 2018016074W WO 2020130166 A1 WO2020130166 A1 WO 2020130166A1
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extract
cells
allergic rhinitis
current
calcium
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PCT/KR2018/016074
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English (en)
Korean (ko)
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김우경
남주현
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동국대학교 산학협력단
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Priority to PCT/KR2018/016074 priority Critical patent/WO2020130166A1/fr
Publication of WO2020130166A1 publication Critical patent/WO2020130166A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/57—Magnoliaceae (Magnolia family)
    • A61K36/575—Magnolia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to an aerosol formulation for inhalation containing a kidney extract that can be effectively used for preventing and treating allergic rhinitis.
  • Allergic rhinitis is a chronic inflammatory disease characterized by a continuous sneezing seizure, a continuous runny nose, and nasal congestion, in which the nasal mucosa is hypersensitive to certain substances (allergens).
  • allergens such as pollen and house dust mites
  • Th2 T cells secrete cytokines such as IL-4 and IL-13, causing class switching, in which B cells secrete IgE, which binds to the Fc receptor of mast cells and mediates IgE. Initiate an allergic reaction.
  • Mast cells with specific IgE binding to specific allergens are degranulated to secrete substances such as histamine that are activated and stored by allergens.
  • the secretion of histamine is combined with histamine receptors expressed in nasal mucosal epithelial cells and blood vessels, causing hypersecretion of mucus, stuffy nose, and the like.
  • the treatment of inflammatory diseases including allergic rhinitis is generally used as an immunosuppressive agent and an antihistamine agent for suppressing the activity of Th2 T cells and mast cells, which can primarily cause allergies.
  • NFAT Nuclear factor of activated T-Cells
  • an antihistamine In the case of an antihistamine, it specifically binds to the histamine receptor expressed on mast cells, and suppresses degranulation so that mast cells cannot be activated and secrete histamine even when exposed to allergens.
  • these effects not only require the use of two drugs, such as anti-inflammatory and anti-histamine, all at once, but also increase the risk of cancer (inflammation inhibitor) and sedation (antihistamine), drowsiness helplessness, and learning ability may decrease. It can cause mental and physical discomfort.
  • intracellular calcium signal generation is important for activation of immune cells and mast cells, and it is known that these pathways that cause intracellular calcium increase are shared with each other.
  • Gq protein-linked receptors are activated to activate PLC (phospholipase C).
  • PLC phospholipase C
  • the activity of these PLCs is IP 3 (inositol-3-phosphate) and DAG (diacylglycerol). Will be created.
  • this increase in intracellular calcium concentration occurs in nasal epithelial cells, which are important for runny nose formation when allergen is exposed.
  • ANO1 a calcium-dependent chlorine ion pathway called ANO1 in response to the increased intracellular calcium ion.
  • ANO1 activation accelerates the production of runny nose, causing the secretion of a clear runny nose that is a characteristic symptom of allergic rhinitis.
  • the present inventor simultaneously inhibits SOCE, TRPV1 and ANO1, which is known to be useful for the prevention and treatment of allergic rhinitis, and inhales for effective allergic rhinitis treatment that can replace the administration of immunosuppressants and antihistamines without side effects.
  • the present invention has been completed by developing an aerosol formulation for use.
  • One object of the present invention is to provide an aerosol formulation for inhalation containing a kidney extract that can be effectively used for preventing and treating allergic rhinitis.
  • One aspect of the present invention provides an aerosol formulation for inhalation for the prevention and treatment of allergic rhinitis containing 0.001% to 0.08% by weight of the Sini extract.
  • the aerosol formulation for inhalation may be one containing 0.01% by weight to 0.08% by weight of the extract.
  • the present invention can be usefully used as a composition for the prevention and treatment of allergic rhinitis diseases, by providing an aerosol preparation for inhalation, which is a parenteral administration method containing a Shini extract in a specific concentration.
  • FIG. 1 is a graph showing the inhibitory effect of calcium ion current (I SOCE ) through ORAI1 of 30% ethanol extract of herbal medicine according to an embodiment of the present invention, ORAI1 ion measured in HEK293-T cells overexpressed with ORAI1 ion channel It shows the patch clamp chart records of 30% ethanol extract 1 mg/ml (red arrow) and ORAI1 inhibitor BTP2 (blue arrow) showing the current of the passage (black arrow) and its inhibitory effect.
  • I SOCE calcium ion current
  • FIG. 2 is a graph showing a current-voltage (I-V) relationship curve of the corresponding arrow shown in FIG. 1.
  • FIG. 4 is a graph showing the inhibitory effect of TRPV1 current, which is a non-selective cation channel of 30% ethanol extract, and the current (black arrow) of TRPV1 ion channel measured in HEK293-T cells overexpressing TRPV1 ion channel and its inhibition Shows the patch clamp chart record of 30 ⁇ g/ml (red arrow), 30 ⁇ g/ml (blue arrow), 100 ⁇ g/ml (blue green arrow) and TRPV1 inhibitor BCTC (gray arrow) of 30% ethanol extract showing the effect.
  • FIG. 5 is a graph showing a current-voltage (I-V) relationship curve of the corresponding arrow shown in FIG. 4.
  • FIG. 6 shows the suppression of T cell proliferation by separating immune cells T cells from PBMC collected from human blood and stimulating them with CD3 and CD28, 30% ethanol extract showing the inhibitory effect of ORAI1 ion current 0.1 It shows the percentage of T cell proliferation inhibition and cell proliferation ratio of mg/ml, 0.3 mg/ml, and 1 mg/ml. Negative refers to T cells that did not stimulate, and Positive refers to cells that stimulated T cells with CD3 and CD28 to induce proliferation.
  • FIG. 7 is a graph showing the inhibitory effect of ANO1 current, which is a calcium-dependent chloride ion channel of 30% ethanol extract, and the current of ANO1 ion channel and HE 30% ethanol extract 0.1 measured in HEK293-T cells overexpressing ANO1 ion channel.
  • Patch clamp chart records of A0-1, mg/ml, 0.3 mg/ml, 1 mg/ml and ANO1 inhibitors are shown.
  • FIG. 8 is a graph showing the current voltage (I-V) relationship curve of FIG. 7.
  • FIG. 10 is a graph showing the current inhibitory effect of the calcium-dependent chloride ion channel (I CaCC ) measured in human airway epithelial cell line Calu-3, an allergen-induced cytokine secreted by airway epithelial cells Calu-3 and Th2 T cells.
  • I CaCC calcium-dependent chloride ion channel
  • FIG. 11 is a graph showing the current inhibitory effect of the calcium-dependent chloride ion channel (I CaCC ) measured in the human airway epithelial cell line Calu-3, 30% ethanol extract from Shin in Calu-3 cells treated with cytokine IL-4 0.1 mg/ml, 0.3 mg/ml, 1 mg/ml of calcium-dependent chloride ion channel current suppression and ANO1 ion channel inhibitor Ani9 current suppression and recorded calcium-dependent chloride ion channel current measured at + 100 mV voltage It shows a summary of the degree of inhibition of the current.
  • I CaCC calcium-dependent chloride ion channel
  • FIG. 12 is a graph showing the therapeutic effect of 30% ethanol extract from Shin in an animal model of allergic rhinitis-induced disease in mice. It shows the average score of clinical symptoms, which is the sum of the sneeze and nose rubbing observed for a minute.
  • FIG. 13 is a graph showing the therapeutic effect of 30% ethanol extract from kidney in animal models of allergic rhinitis-induced disease in mice. Cyto secreted from spleen cells collected from animal models of kidney treated by concentration of 30% ethanol extract Cain IL-4.
  • FIG. 14 is a graph showing the therapeutic effect of 30% ethanol extract from kidney in the animal model of allergic rhinitis-induced disease in mice, and the cytokine secreted from spleen cells collected from the animal model in which the kidney was treated by concentration of 30% ethanol extract Cain IL-13.
  • the present invention provides an aerosol formulation for inhalation for the prevention and treatment of allergic rhinitis, which contains 0.001 to 0.08% by weight of the Sini extract.
  • the present inventors studied the pharmacological mechanism of Shini extract, and then inhibited the action of calcium ion current (I SOCE ) through ORAI1, inhibition of TRPV1 current, which is a non-selective cation pathway, and inhibition of ANO1 current, which is a calcium-dependent chloride ion channel. By this, it was confirmed that the extract has an effect on preventing and treating allergic rhinitis.
  • the present invention is the most effective at concentrations containing 0.001% by weight (0.01 mg/ml) to 0.08% by weight (0.8 mg/ml) of Shini extract after studying several pharmacological mechanisms for preventing and treating allergic rhinitis of the Sini extract. Was confirmed.
  • the aerosol formulation for inhalation used in the present invention contains 0.001 to 0.08% by weight of the extract of Shini, and preferably 0.01 to 0.08% by weight. If the extract of Shini is administered at a concentration of less than 0.001% by weight, it is difficult to expect a preventive and therapeutic effect against allergic rhinitis, and surprisingly, even if the Sini extract is administered at a concentration of 0.08% by weight (0.8 mg/ml) or more, It has no preventive and therapeutic effect.
  • the present invention is the most effective at concentrations containing 0.0001% by weight (0.001 mg/ml) to 0.008% by weight (0.08mg/ml) of Shini extract after studying several pharmacological mechanisms for preventing and treating allergic rhinitis of the Sini extract was confirmed.
  • the aerosol formulation for inhalation used in the present invention contains 0.001 to 0.08% by weight of the extract of Shini, and preferably 0.01 to 0.08% by weight. If the extract of Shini is administered at a concentration of less than 0.001% by weight, it is difficult to expect a preventive and therapeutic effect against allergic rhinitis, and surprisingly, even if the Sini extract is administered at a concentration of 0.08% by weight (0.8 mg/ml) or more, It has no preventive and therapeutic effect.
  • the present invention can be administered by inhaling the Shini extract contained in the aerosol formulation for inhalation once to several times a day.
  • Shinyi extract used in the present invention can be prepared by the following method. That is, after removing the foreign matter by washing the water with water, it is dried and crushed in the shade. Shini can be used without restrictions such as those grown or commercially available. An appropriate amount of solvent is added to the powder of the crushed kidney so that it is completely immersed.
  • Shini's mixed powder can be extracted using a conventional extraction solvent, preferably, (a) anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (eg methanol, ethanol, propanol, butanol, normal-propanol, iso -Propanol and normal-butanol, etc.), (b) a mixed solvent of the lower alcohol and water, (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, ( g) Hexane, (h) diethyl ether, (i) butyl acetate or (j) can be extracted with water, and the ethanol solution used as a solvent is extracted with ethanol 30: distilled water 70 in terms of extraction yield.
  • a conventional extraction solvent preferably, (a) anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (eg methanol, ethanol, propanol, butanol, normal-
  • the extract When extracting the medicine with ethanol, it is preferable to extract by heating with a heavy bath for 3 hours, and ethanol may be added at 5 to 20 parts by weight per 1 part by weight.
  • the extract may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.
  • the extract may be extracted with any one or more solvents selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixed solvents thereof.
  • the extract may be extracted by using a mixed solvent in which the weight ratio of ethanol and water is 3:10 to 6:10.
  • the composition may include as much as 0.001% to 0.08% by weight of the Shini extract.
  • the present invention is to provide an aerosol formulation for inhalation containing the extract of the kidney.
  • the carrier contained in the aerosol formulation for inhalation of the present invention is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto. .
  • the pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, etc.
  • the aerosol formulation for inhalation of the present invention may include various bases and/or additives necessary and appropriate for the formulation of the formulation, and non-ionic surfactants, silicone polymers, constitution pigments, and the like within the scope of not impairing the effect.
  • the dried Shini was purchased and the powder was prepared using a grinder. Shini powder 200g in 2kg of 30% ethanol, and stirred for 3 hours by heating with a heavy bath, Whatman No. 1
  • the extraction solution was filtered using filter paper. Thereafter, the supernatant was taken using a centrifuge, and the remaining residue was extracted twice by the same method.
  • the filtered extract solution was freeze-dried to recover the dried powder of ethanol extract from Shin.
  • HEK293T cells (catalog no. CRL-3216) were used for pre-sale in the American type culture collection (ATCC, Manassas, VA, USA). The current measured was amplified using an Axopatch 200B (Molecular Device) amplifier, digitized through a Digidata 1440A (Molecular Device), and recorded with pClamp 10.4 (Molecular Device).
  • the voltage was fixed at -100 mV, and the inclined voltage was changed from -130 mV to -70 mV at intervals of 20 seconds for 100 ms.
  • Inositole was added to the intracellular solution to activate SOCE.
  • -3-phosphate (IP 3 ) was added.
  • Example 1 In order to confirm whether the Shini extract prepared in Example 1 can inhibit TRPV1, which is one of the calcium channels, a whole cell patch clamp was performed using a HEK293T cell line overexpressed with human TRPV1 protein.
  • I TRPV1 ion current through TRPV1 is increased by treating 1 uM of capsaicin capable of activating TRPV1, 10, 30, and 100 ug/ml of Shini extract were sequentially processed.
  • PBMC Peripheral Blood Mononuclear cell
  • T cells were separated from the separated PBMC using a Pan T cell isolation Kit (Milteny biotech). The separation process was performed according to the method provided by the manufacturer.
  • T cells In order to confirm the proliferation of the isolated T cells, 2 ⁇ M of a fluorescence-indicating substance, carboxy-fluorescein succinimidyl ester (CFSE), was added to T cells and incubated at room temperature for 10 minutes.
  • the labeled T cells were dispensed into a 96 well plate to be 2 ⁇ 10 5 cells/well, and 3 ⁇ m/ml Anti-Human CD3 and 2 ⁇ g/ml Anti-Human CD28 were added to RPMI 1640. Cultured daily. After 3 days, only CD4 + T cells were stained using Anti-Human CD4-APC (BD Pharmingen), and then cell proliferation was analyzed using FACS.
  • CFSE carboxy-fluorescein succinimidyl ester
  • Sini extract inhibits the proliferation of human T cells in a concentration-dependent manner.
  • the ANO1 ion channel which is a calcium-dependent chlorine ion channel, is a very important ion channel for the secretion of mucus and electrolytes from the nasal epithelial cells, that is, the formation of a runny nose, so that the human ANO1 protein is used to determine how the new extract affects the human ANO1 ion channel.
  • Whole cell patch clamp was performed using the overexpressed HEK293T cell line.
  • Example 6 Calcium-dependent chlorine ion channel activity inhibition of Sini extract in human airway epithelial cells
  • Human airway epithelial cells have various chlorine ion channels including ANO1 ion channels, so IL-4, a cytokine secreted by Th2 T cells using human airway epithelial cell line Calu-3 (ATCC), is involved in runny nose production. It was confirmed whether the expression of the chlorine ion channel is increased and whether the extract of Shin can effectively suppress this.
  • a female 7-week-old Balb/c mouse was purchased (Orient Bio, Gyeonggi-do, Korea), reared under free-feeding conditions, stabilized in a breeding room for one week, and used in the experiment.
  • Ovalbumin OVA, sigma A5503
  • Inject Alum Alum, Thermo scientific Prod#77161
  • Shini extract and hydrocortisone Hydrocortisone, sigma H0888
  • 0.2 ml of the antigen was injected intraperitoneally once a week for 3 weeks.
  • the normal control (NL) was injected with 200 ⁇ l of DPBS containing 2 mg of Alum
  • the positive control (PC) and experimental group were injected with 25 ⁇ g of OVA and 200 ⁇ l of DPBS containing Alum 2 mg.
  • nasal drops were applied once a day for 8 days.
  • 30 ⁇ l of DPBS as a normal control group and 30 ⁇ l of DPBS including 100 ⁇ g of OVA were added to the nasal cavity in the positive control group and the experimental group.
  • DPBS contains 30% ethanol extracts of 0.001 mg/ml, 0.01 mg/ml, 0.1 mg/ml, 0.3 mg/ml, 0.8 mg/ml, 1 mg/ml, 3 mg/ml and 10 mg/ml, respectively. 20 ⁇ l of the solution was added to the nasal cavity once, and Hydrocortisone was added to the nasal cavity with 1 mg/ml included in 20 ⁇ l of DPBS.
  • OVA 1 mg/ml was added to 5 x 10 6 cells/ml of spleen cells, and cultured at 37.5° C. for 72 hours, followed by supernatant of the culture.
  • concentration of IL-4 was measured using Magnetic Luminex Assay (R&D Systems ® , Minneapolis, MN, USA) according to the manufacturer's protocol (Cytokine measurements).

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Abstract

La présente invention concerne une préparation d'aérosol pour inhalation comprenant un extrait de Magnolia flos, qui peut être efficacement utilisée pour prévenir et traiter la rhinite allergique. La présente invention concerne une préparation d'aérosol comprenant une concentration particulière en un extrait de Magnolia flos, qui est administrée par voie parentérale par inhalation et, ainsi, peut être avantageusement utilisée en tant que composition pour la prévention et le traitement d'une maladie de type rhinite allergique.
PCT/KR2018/016074 2018-12-18 2018-12-18 Composition comprenant un extrait de magnolia flos pour la prévention et le traitement de la rhinite allergique WO2020130166A1 (fr)

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PCT/KR2018/016074 WO2020130166A1 (fr) 2018-12-18 2018-12-18 Composition comprenant un extrait de magnolia flos pour la prévention et le traitement de la rhinite allergique

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20000026053A (ko) * 1998-10-17 2000-05-06 박호군 류코트리엔 생성 저해활성을 가지는 신이 추출물 및 신이로부터분리한 리그난 화합물
JP2004026813A (ja) * 2002-05-07 2004-01-29 Taisho Pharmaceut Co Ltd 医薬組成物
KR100735541B1 (ko) * 2005-09-26 2007-07-06 우석대학교 산학협력단 비강세척제 조성물 및 그 제조방법
JP2010047566A (ja) * 2008-07-23 2010-03-04 Takeda Chem Ind Ltd 医薬組成物
KR20140072486A (ko) * 2012-12-05 2014-06-13 구미경 생약 혼합물의 추출물을 함유하는 축농증 또는 알레르기성 비염의 예방 또는 치료용 조성물
KR20190058372A (ko) * 2017-11-21 2019-05-29 동국대학교 산학협력단 신이 추출물을 포함하는 알레르기 비염 예방 및 치료용 조성물

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20000026053A (ko) * 1998-10-17 2000-05-06 박호군 류코트리엔 생성 저해활성을 가지는 신이 추출물 및 신이로부터분리한 리그난 화합물
JP2004026813A (ja) * 2002-05-07 2004-01-29 Taisho Pharmaceut Co Ltd 医薬組成物
KR100735541B1 (ko) * 2005-09-26 2007-07-06 우석대학교 산학협력단 비강세척제 조성물 및 그 제조방법
JP2010047566A (ja) * 2008-07-23 2010-03-04 Takeda Chem Ind Ltd 医薬組成物
KR20140072486A (ko) * 2012-12-05 2014-06-13 구미경 생약 혼합물의 추출물을 함유하는 축농증 또는 알레르기성 비염의 예방 또는 치료용 조성물
KR20190058372A (ko) * 2017-11-21 2019-05-29 동국대학교 산학협력단 신이 추출물을 포함하는 알레르기 비염 예방 및 치료용 조성물

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